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1.  Catalytic surface radical in dye-decolorizing peroxidase: a computational, spectroscopic and site-directed mutagenesis study 
Biochemical Journal  2015;466(Pt 2):253-262.
Dye-decolorizing peroxidase (DyP) of Auricularia auricula-judae has been expressed in Escherichia coli as a representative of a new DyP family, and subjected to mutagenic, spectroscopic, crystallographic and computational studies. The crystal structure of DyP shows a buried haem cofactor, and surface tryptophan and tyrosine residues potentially involved in long-range electron transfer from bulky dyes. Simulations using PELE (Protein Energy Landscape Exploration) software provided several binding-energy optima for the anthraquinone-type RB19 (Reactive Blue 19) near the above aromatic residues and the haem access-channel. Subsequent QM/MM (quantum mechanics/molecular mechanics) calculations showed a higher tendency of Trp-377 than other exposed haem-neighbouring residues to harbour a catalytic protein radical, and identified the electron-transfer pathway. The existence of such a radical in H2O2-activated DyP was shown by low-temperature EPR, being identified as a mixed tryptophanyl/tyrosyl radical in multifrequency experiments. The signal was dominated by the Trp-377 neutral radical contribution, which disappeared in the W377S variant, and included a tyrosyl contribution assigned to Tyr-337 after analysing the W377S spectra. Kinetics of substrate oxidation by DyP suggests the existence of high- and low-turnover sites. The high-turnover site for oxidation of RB19 (kcat> 200 s−1) and other DyP substrates was assigned to Trp-377 since it was absent from the W377S variant. The low-turnover site/s (RB19 kcat ~20 s−1) could correspond to the haem access-channel, since activity was decreased when the haem channel was occluded by the G169L mutation. If a tyrosine residue is also involved, it will be different from Tyr-337 since all activities are largely unaffected in the Y337S variant.
We demonstrate that an exposed tryptophan is responsible for high-turnover oxidation by DyP, a representative of a new protein superfamily. Long-range electron transfer from surface tryptophan residues forming radicals appears as a general mechanism for peroxidase oxidation of bulky substrates.
doi:10.1042/BJ20141211
PMCID: PMC4357238  PMID: 25495127
catalytic protein radical; dye-decolorizing peroxidase; EPR spectroscopy; molecular docking; QM/MM; site-directed mutagenesis; ABTS, 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid); DMP, 2,6-dimethoxyphenol; DyP, dye-decolorizing peroxidase; hfcc, hyperfine coupling constant; LiP, lignin peroxidase; LRET, long-range electron transfer; MM, molecular mechanics; NBS, N-bromosuccinimide; PELE, Protein Energy Landscape Exploration; QM, quantum mechanics; RB5, Reactive Black 5; RB19, Reactive Blue 19; TNM, tetranitromethane; VA, veratryl alcohol; VP, versatile peroxidase; WT, wild-type
2.  Crystallization and preliminary X-ray diffraction analysis of the fructofuranosidase from Xanthophyllomyces dendrorhous  
The invertase from X. dendrorhous has been purified, deglycosylated and crystallized and diffraction data have been collected to 2.3 Å resolution.
Xanthophyllomyces dendrorhous invertase is an extracellular enzyme that releases β-fructose from the nonreducing termini of various β-d-fructofuranoside substrates. Its ability to produce neokestose by transglycosylation makes this enzyme an interesting research target for applications in industrial biotechnology. The native enzyme, which is highly glycosylated, failed to crystallize. Therefore, it was submitted to EndoH deglycosylating treatment and crystals were grown by vapour-diffusion methods. The crystals belonged to space group P21212, with unit-cell parameters a = 75.29, b = 204.93, c = 146.25 Å. Several diffraction data sets were collected using a synchrotron source. Self-rotation function and gel-filtration experiments suggested that the enzyme is a dimer with twofold symmetry.
doi:10.1107/S1744309110029192
PMCID: PMC3001643  PMID: 21045290
yeast invertase; β-fructofuranosidases; glycoside hydrolase family 32
3.  Molecular and Biochemical Characterization of a β-Fructofuranosidase from Xanthophyllomyces dendrorhous▿ †  
An extracellular β-fructofuranosidase from the yeast Xanthophyllomyces dendrorhous was characterized biochemically, molecularly, and phylogenetically. This enzyme is a glycoprotein with an estimated molecular mass of 160 kDa, of which the N-linked carbohydrate accounts for 60% of the total mass. It displays optimum activity at pH 5.0 to 6.5, and its thermophilicity (with maximum activity at 65 to 70°C) and thermostability (with a T50 in the range 66 to 71°C) is higher than that exhibited by most yeast invertases. The enzyme was able to hydrolyze fructosyl-β-(2→1)-linked carbohydrates such as sucrose, 1-kestose, or nystose, although its catalytic efficiency, defined by the kcat/Km ratio, indicates that it hydrolyzes sucrose approximately 4.2 times more efficiently than 1-kestose. Unlike other microbial β-fructofuranosidases, the enzyme from X. dendrorhous produces neokestose as the main transglycosylation product, a potentially novel bifidogenic trisaccharide. Using a 41% (wt/vol) sucrose solution, the maximum fructooligosaccharide concentration reached was 65.9 g liter−1. In addition, we isolated and sequenced the X. dendrorhous β-fructofuranosidase gene (Xd-INV), showing that it encodes a putative mature polypeptide of 595 amino acids and that it shares significant identity with other fungal, yeast, and plant β-fructofuranosidases, all members of family 32 of the glycosyl-hydrolases. We demonstrate that the Xd-INV could functionally complement the suc2 mutation of Saccharomyces cerevisiae and, finally, a structural model of the new enzyme based on the homologous invertase from Arabidopsis thaliana has also been obtained.
doi:10.1128/AEM.02061-08
PMCID: PMC2643559  PMID: 19088319

Results 1-3 (3)